EP2532744A1 - Glycerin-3-phosphatacyltransferase-homolog und seine verwendung - Google Patents

Glycerin-3-phosphatacyltransferase-homolog und seine verwendung Download PDF

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EP2532744A1
EP2532744A1 EP11739829A EP11739829A EP2532744A1 EP 2532744 A1 EP2532744 A1 EP 2532744A1 EP 11739829 A EP11739829 A EP 11739829A EP 11739829 A EP11739829 A EP 11739829A EP 2532744 A1 EP2532744 A1 EP 2532744A1
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seq
protein
activity
expressing
host
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EP2532744B1 (de
EP2532744A4 (de
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Misa Ochiai
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Suntory Holdings Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01015—Glycerol-3-phosphate O-acyltransferase (2.3.1.15)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Definitions

  • the present invention relates to a novel acyltransferase gene and use thereof.
  • the acyltransferase gene of the present invention may be a glycerol 3-phosphate acyltransferase (GPAT) gene and/or a glycerone phosphate O-acyltransferase (GNPAT) gene.
  • GPAT glycerol 3-phosphate acyltransferase
  • GPAT glycerone phosphate O-acyltransferase
  • Fatty acids are important components constituting lipids such as phospholipid and triacylglycerol. Fatty acids having two or more unsaturated bond sites are collectively called polyunsaturated fatty acids (PUFAs). Specifically, for example, arachidonic acid, dihomo- ⁇ -linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid are known, and various bioactivities thereof have been reported (Non-Patent Literature 1). Some of the polyunsaturated fatty acids cannot be synthesized in animal bodies, and such polyunsaturated fatty acids should be ingested from foods as essential fatty acids.
  • PUFAs polyunsaturated fatty acids
  • the polyunsaturated fatty acids are contained in various organs and tissues.
  • arachidonic acid is isolated from lipids extracted from suprarenal gland and liver of animals.
  • the amounts of these polyunsaturated fatty acids contained in animal organs are, however, small, and the polyunsaturated fatty acids extracted and isolated from animal organs only are insufficient for a large amount of supply thereof.
  • microbial techniques have been developed for obtaining polyunsaturated fatty acids by culturing various microorganisms.
  • microorganisms in the genera Mortierella are known to efficiently produce lipids containing polyunsaturated fatty acids such as arachidonic acid.
  • Other attempts have also been made to produce polyunsaturated fatty acids in plants.
  • Polyunsaturated fatty acids are known to constitute reserve lipids such as triacylglycerol and accumulate within microorganism cells or plant seeds.
  • Triacylglycerol as a reserve lipid is generated in living bodies as follows: An acyl group is transferred to glycerol 3-phosphate by glycerol 3-phosphate acyltransferase to generate lysophosphatidic acid. Another acyl group is transferred to the lysophosphatidic acid by lysophosphatidic acid acyltransferase to generate phosphatidic acid. The phosphatidic acid is dephosphorylated by phosphatidic acid phosphatase to generate diacylglycerol. A further acyl group is transferred to the diacylglycerol by diacylglycerol acyltransferase to ultimately generate triacylglycerol.
  • glycerol 3-phosphate acyltransferase (hereinafter, also referred to as "GPAT": EC 2.3.1.15) involves a reaction generating lysophosphatidic acid through acylation of glycerol 3-phosphate.
  • Non-Patent Literature 2 GPAT genes derived from mammals
  • GPAT genes derived from plants two types of GPAT genes, i.e., a microsomal type (membrane-bound form) and a mitochondrial type (membrane-bound form)
  • GPAT genes derived from plants three types of GPAT genes, i.e., a microsomal type (membrane-bound form), a mitochondrial type (membrane-bound form), and a chloroplast type (free form), have been cloned (Non-Patent Literature 3).
  • GPAT genes derived from fungi, Saccharomyces cerevisiae two types of GPAT genes, i.e., microsomal type (membrane-bound form) GPT2/GAT1 (YKR067w) and SCT1/GAT2 (YBL011w), have been cloned, and it is known that simultaneous deletion of these types of GPAT genes results in death (Non-Patent Literature 4).
  • GPT2 has an activity showing broad substrate specificity to fatty acids from palmitic acid (16:0) to oleic acid (18:1)
  • SCT1 shows high substrate selectivity to fatty acids having 16 carbon atoms such as palmitic acid (16:0) and palmitoleic acid (16:1)
  • GPAT gene has been cloned from various biological species.
  • GPAT derived from a lipid-producing fungus, the genera Mortierella is reported as follows.
  • Non-Patent Literature 6 GPAT derived from Mortierella ramanniana .
  • Mortierella alpina hereinafter, also referred to as "M. alpina”
  • M. alpina it has been reported that a glycerol 3-phosphate acyltransferase activity resides in a microsomal fraction
  • MaGPAT1 GPAT cloned from M. alpina (ATCC No. 16266) (hereinafter, referred to as MaGPAT1 (ATCC No. 16266)) was expressed in Yarrowia lipolytica that had been transformed such that eicosapentaenoic acid (EPA) can be biosynthesized, and as a result, a proportion of dihomo- ⁇ -linolenic acid (DGLA) (20:3) increased, whereas a proportion of oleic acid (18:1) decreased, among the total fatty acids.
  • DGLA dihomo- ⁇ -linolenic acid
  • MaGPAT2 a GPAT homolog, MaGPAT2
  • strain 1S-4 strain 1S-4
  • the homolog has a substrate specificity different from that of MaGPAT1 (Patent Literature 3). That is, when they are expressed in yeast, MaGPAT1 increases the content of palmitic acid in the lipid produced by the yeast, whereas MaGPAT2 increases the content of oleic acid in the lipid produced by the yeast.
  • GPAT genes When the previously reported GPAT genes are introduced into host cells and are expressed therein, a fatty acid composition produced by the host is restricted by their substrate specificity. Identification of a novel gene that can produce an intended fatty acid composition by introduction or expression in a host cell is required.
  • a protein and a nucleic acid that can achieve production of a fat having an intended compositional ratio of fatty acids, can increase the content of an intended fatty acid, or can increase the amount of a reserve lipid, triacylglycerol (TG), through expression or introduction in the host cells.
  • TG triacylglycerol
  • the present inventor has diligently studied to solve the above-mentioned problems.
  • the inventor has analyzed the genome of a lipid-producing fungus, Mortierella alpina, and extracted sequences having a high dgree of homology with known glycerol 3-phosphate acyltransferase (GPAT) genes from the genome. Further, in order to obtain a full-length of the open reading frame (ORF) encoding GPAT, a full-length cDNA was cloned by screening or PCR of a cDNA library.
  • ORF open reading frame
  • the present inventor has tried producing a fatty acid composition by introducing the gene into host cells having high proliferative ability, such as yeast, and as a result, the inventor has successfully cloned a gene related to a novel GPAT that has a different substrate specificity and can generate a fatty acid composition different from the fatty acid composition produced by the host cells expressing conventional GPAT, and the present invention has been accomplished. That is, the present invention is as follows.
  • nucleic acid according to aspect (1) wherein the nucleic acid is any one selected from (a) to (g) below:
  • nucleic acid according to aspect (4) wherein the nucleic acid is any one selected from (a) to (g) below:
  • a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9.
  • a recombinant vector comprising the nucleic acid according to any one of aspects (1) to (5).
  • a fatty acid composition comprising a fatty acid or a lipid obtainable by culturing the transformant according to aspect (12).
  • a method of producing a fatty acid composition comprising collecting a fatty acid or a lipid from a culture obtained by culturing the transformant according to aspect (12).
  • a food comprising the fatty acid composition according to aspect (13).
  • the GPAT of the present invention has substrate specificity different from that of a conventional GPAT and can allow a host to produce fatty acids having a composition different from that of fatty acids produced by a host expressing a conventional GPAT. This can provide lipids having intended characteristics and effects and is therefore useful in application to foods, cosmetics, pharmaceuticals, soap, etc.
  • the GPAT of the present invention can enhance the producibility of fatty acids and reserve lipids and thus can enhance the productivity of polyunsaturated fatty acids in microorganisms and plants, and is preferable.
  • the present invention relates to a novel acyltransferase gene derived from Mortierella and use thereof.
  • the acyltransferase of the present invention may be an acyltransferase that acylates glycerol 3-phosphate to generate lysophosphatidic acid and/or that transfers an acyl group to a hydroxyl group of glycerone phosphate.
  • the acyltransferase of the present invention is an enzyme that catalyzes a transfer reaction of an acyl group to glycerol 3-phosphate and/or glycerone phosphate.
  • the acyl-group receptor for the enzyme of the present invention is usually glycerol 3-phosphate and/or glycerone phosphate, but is not limited thereto.
  • the acyltransferase of the present invention may have an activity as a glycerol 3-phosphate acyltransferase (GPAT) and/or a glycerone phosphate O-acyltransferase (GNPAT).
  • GPAT glycerol 3-phosphate acyltransferase
  • GPAT glycerone phosphate O-acyltransferase
  • the enzyme of the present invention may also be conveniently referred to as "glycerol 3-phosphate acyltransferase” or "GPAT" regardless of its actual activity.
  • Examples of glycerol 3-phosphate acyltransferase (GPAT) of the present invention encompass MaGPAT4, MaGPAT4-long, and MaGPAT5.
  • the correspondence between cDNA, CDS, and ORF encoding MaGPAT4, MaGPAT4-long, or MaGPAT5, and a deduced amino acid sequence thereof is summarized in Table 1.
  • Sequences related to MaGPAT4 of the present invention include SEQ ID NO: 2 showing the amino acid sequence of MaGPAT4; SEQ ID NO: 1 showing the sequence of the ORF region of MaGPAT4; and SEQ ID NO: 3 showing the sequence of the CDS or cDNA of MaGPAT4.
  • SEQ ID NO: 1 corresponds to the nucleotides 1 to 2475 in the sequence set forth in SEQ ID NO: 3.
  • Sequences related to MaGPAT4-long of the present invention include SEQ ID NO: 5 showing the amino acid sequence of MaGPAT4-long; SEQ ID NO: 4 showing the sequence of the ORF region of MaGPAT4-long; and SEQ ID NO: 6 showing the sequence of the CDS or cDNA region of MaGPAT4-long.
  • SEQ ID NO: 1 corresponds to the nucleotides 1 to 2475 in the sequence set forth in SEQ ID NO: 3.
  • SEQ ID NO: 4 corresponds to the nucleotides 1 to 2643 in the sequence set forth in SEQ ID NO: 6.
  • the amino acid sequence and the nucleotide sequence of MaGPAT4 constitute parts of the amino acid sequence and the nucleotide sequence of MaGPAT4-long, respectively.
  • SEQ ID NO: 7 shows a genomic nucleotide sequence encoding MaGPAT4 and MAGPAT4-long of the present invention.
  • the genomic sequence set forth in SEQ ID NO: 7 is composed of ten exons and nine introns, and the exon regions correspond to the nucleotides 596 to 744, 850 to 924,1302 to 1396, 1480 to 1726, 1854 to 2279, 2370 to 2632, 2724 to 3299, 3390 to 3471, 3575 to 4024, and 4133 to 4248 in SEQ ID NO: 7.
  • the genomic sequence set forth in SEQ ID NO: 7 is composed of ten exons and nine introns, and the exon regions correspond to the nucleotides 428 to 744, 850 to 924,1302 to 1396, 1480 to 1726, 1854 to 2279, 2370 to 2632, 2724 to 3299, 3390 to 3471, 3575 to 4024, and 4133 to 4248 in SEQ ID NO: 7.
  • Sequences related to MaGPAT5 of the present invention include SEQ ID NO: 9 showing the amino acid sequence of MaGPAT5; SEQ ID NO: 8 showing the sequence of the ORF region of MaGPAT5; SEQ ID NO: 10 showing the sequence of the CDS region of MaGPAT5; and SEQ ID NO: 11 showing the sequence of the cDNA for MaGPAT5.
  • SEQ ID NO: 10 corresponds to the nucleotides 225 to 2591 in the sequence set forth in SEQ ID NO: 11; and SEQ ID NO: 8 corresponds to the nucleotides 225 to 2588 in the sequence set forth in SEQ ID NO: 11 and the nucleotides 1 to 2364 in the sequence set forth in SEQ ID NO: 10.
  • SEQ ID NO: 12 shows a genomic nucleotide sequence encoding MaGPAT5 of the present invention.
  • the genomic sequence set forth in SEQ ID NO: 12 is composed of three exons and two introns, and the exon regions correspond to the nucleotides 1 to 302, 457 to 1676, and 1754 to 2598 in SEQ ID NO: 12.
  • the nucleic acids of the present invention encompass single-stranded and double-stranded DNAs and also their complementary RNAs, which may be either naturally occurring or artificially prepared.
  • DNA include, but not limited to, genomic DNAs, cDNAs corresponding to the genomic DNAs, chemically synthesized DNAs, PCR-amplified DNAs, combinations thereof, and DNA/RNA hybrids.
  • nucleic acids of the present invention include (a) nucleic acids containing the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8, (b) nucleic acids containing a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9, (c) nucleic acids containing the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 11, and (d) nucleic acids containing the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12.
  • nucleotide sequence data of ESTs or genomic DNAs from organisms having GPAT activity may be used to search for a nucleotide sequence encoding a protein having a high identity with known proteins having GPAT activity.
  • Preferred organisms having GPAT activity are lipid-producing fungi including, but not limited to, M. alpina.
  • a cDNA library is first prepared.
  • the cDNA library may be prepared by referring to " Molecular Cloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001 )).
  • a commercially available cDNA library preparation kit may be used.
  • Examples of a method of preparing a cDNA library suitable for the present invention are as follows. That is, an appropriate strain of M. alpine, a lipid-producing fungus, is inoculated into an appropriate medium and is pre-cultured for an appropriate period. Culture conditions suitable for this pre-culture are, for example, a medium composition of 1.8% glucose and 1% yeast extract, pH 6.0, a culture period of 3 to 4 days, and a culture temperature of 28°C.
  • a medium composition suitable for the main culture is, for example, 1.8% glucose, 1% soybean powder, 0.1% olive oil, 0.01% Adekanol, 0.3% KH 2 PO 4 , 0.1% Na2S04,0-05% CaCl 2 ⁇ 2H 2 O, and 0.05% MgCl 2 ⁇ 6H 2 O, and pH 6.0.
  • Culture conditions suitable for the main culture are, for example, aeration and agitation culture at 300 rpm, 1 vvm, and 26°C for 8 days. An appropriate amount of glucose may be added during culture.
  • the cultured product is sampled at appropriate time points during the main culture, from which the cells are collected to prepare total RNA.
  • the total RNA may be prepared by any known method such as a guanidine hydrochloride/CsCl method. From the resulting total RNA, poly(A) + RNA can be purified using a commercially available kit, and a cDNA library can be prepared using a commercially available kit. The nucleotide sequence of any clone from the prepared cDNA library is determined using primers that are designed on a vector to allow determination of the nucleotide sequence of an insert. As a result, ESTs can be obtained. For example, when a ZAP-cDNA GigapackIII Gold Cloning Kit (Stratagene) is used for preparing a cDNA library, directional cloning is possible.
  • ZAP-cDNA GigapackIII Gold Cloning Kit (Stratagene)
  • genomic DNA In analysis of genomic DNA, cells of an organism having GPAT activity are cultured, and genomic DNA is prepared from the cells. The nucleotide sequence of the resulting genomic DNA is determined, and the determined nucleotide sequence is assembled. From the finally obtained supercontig sequence, a sequence encoding an amino acid sequence having a high homology with the amino acid sequence of a known protein having GPAT activity is searched. From the supercontig sequence giving a hit as that encoding such an amino acid sequence, primers are prepared. PCR is performed using the cDNA library as a template, and the resulting DNA fragment is inserted into a plasmid for cloning. PCR is performed using the cloned plasmid as a template and the above-mentioned primers to prepare a probe. The cDNA library is screened using the resulting probe.
  • a homology search of deduced amino acid sequences of MaGPAT4 and MaGPAT5 of the present invention was performed with BLASTp program against amino acid sequences registered in GenBank.
  • An amino acid sequence having a high identity with that of MaGPAT4 is an amino acid sequence (GenBank accession No. XP_001224211) of a presumed protein derived from ascomycete Chaetomium globosum CBS 148.51, and the identity is 39.3%.
  • the amino acid sequence of MaGPAT4 also has a homology with glycerone phosphate O-acyltransferase (GNPAT; GenBank accession No. AAH00450) derived from human being, and the amino acid identity is 22.6%.
  • MaGPAT4 and plsB protein which is GPAT derived from Escherichia coli ( E. coli )
  • An amino acid sequence having a high identity with that of MaGPAT5 is an amino acid sequence (GenBank accession No. XP_759516) of a presumed protein derived from basidiomycete Ustilago maydis 521, and the identity is 15.4%.
  • the present invention also encompasses nucleic acids functionally equivalent to a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 (hereinafter also referred to as “the nucleotide sequence of the present invention") or a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 (hereinafter also referred to as "the amino acid sequence of the present invention”).
  • the term “functionally equivalent” refers to that a protein encoded by the nucleotide sequence of the present invention and a protein consisting of the amino acid sequence of the present invention have a glycerol 3-phosphate acyltransferase (GPAT) activity and/or a glycerone phosphate O-acyltransferase (GNPAT) activity.
  • GPAT glycerol 3-phosphate acyltransferase
  • GPAT glycerone phosphate O-acyltransferase
  • the term “functionally equivalent” may refer to exsitense of any one of the following activities, in regard of a compositional ratio of fatty acids in a host expressing a protein encoded by a nucleotide sequence of the present invention or a protein consisting of an amino acid sequence of the present invention,:
  • nucleic acid comprising a nucleotide sequence encoding a protein that consists of an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and has the activity of the present invention
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences encoding a protein that consists of an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and has the activity of the present invention.
  • nucleotide sequence contained in the nucleic acid of the present invention is a nucleotide sequence encoding a protein having the above-described activity of the present invention and consisting of:
  • substitution is preferably conservative, which means replacement of a certain amino acid residue by another residue having similar physical and chemical characteristics. It may be any substitution that does not substantially alter the structural characteristics of the original sequence. For example, any substitution is possible as long as the substituted amino acids do not disrupt the helix of the original sequence or do not disrupt any other type of secondary structure characterizing the original sequence.
  • substituents may include an unnatural amino acid residue, a peptidomimetic, or a reversed or inverted form where an unsubstituted region is reversed or inverted in the amino acid sequence.
  • a member of one of the above groups may be replaced by a member from another group.
  • the hydropathic indices of amino acids (hydropathic amino acid indices) ( Kyte, et al., J. Mol. Biol., 157: 105-131 (1982 )) are preferably considered.
  • amino acid substitutions may be accomplished on the basis of hydrophilicity.
  • amino acid residues corresponding to the 316th, 319th, and 351st amino acids in SEQ ID NO: 2 are desirably glycine, serine, and proline, respectively.
  • amino acid residues corresponding to the 430th, 432nd, and 465th amino acids are desirably glycine, serine, and proline, respectively.
  • nucleotides, amino acids, and abbreviations thereof are those according to the IUPAC-IUB Commission on Biochemical Nomenclature or those conventionally used in the art, for example, as described in Immunology--A Synthesis (second edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Massachusetts (1991 )).
  • amino acids which may have optical isomers are intended to represent their L-isomers, unless otherwise specified.
  • Stereoisomers such as D-amino acids of the above-mentioned amino acids, unnatural amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids can be also members constituting the proteins of the present invention.
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard usage and convention in the art.
  • the left-hand end of single-stranded polynucleotide sequences is the 5'-end and the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5'-direction.
  • Those skilled in the art can design and prepare appropriate mutants of the proteins described in the specification using techniques known in the art. For example, they can identify a region in a protein molecule which the region is suitable for changing the structure of the protein of the present invention without impairing the biological activity of the protein by targeting a region which appears to be less important for the biological activity of the protein. Those skilled in the art also can identify a residue or region conserved between similar proteins. Those skilled in the art also can introduce conservative amino acid substitution into a region that appears to be important for the biological activity or structure of the protein of the present invention, without impairing the biological activity and without adversely affecting the polypeptide structure of the protein.
  • Those skilled in the art can conduct a so-called structure-function study, which identifies residues of a peptide that is similar to a peptide of a protein of the present invention and important for a biological activity or structure of the protein, compares the amino acid residues of these two peptides, and thereby predicts which residue in the protein similar to the protein of the present invention is the amino acid residue corresponding to the important amino acid residue for the biological activity or structure. They also can select a mutant which maintains the biological activity of the protein of the present invention by selecting an amino acid substituent chemically similar to the thus predicted amino acid residue. Further, those skilled in the art can analyze the three-dimensional structure and amino acid sequence of this protein mutant.
  • those skilled in the art can predict an alignment of amino acid residues involved in the three-dimensional structure of the protein based on the analytical results thus obtained. Though amino acid residues predicted to be on the protein surface may be involved in important interaction with other molecules, those skilled in the art would be able to prepare a mutant that causes no change in these amino acid residues predicted to be on the protein surface, on the basis of analytical results as mentioned above. Those skilled in the art can also prepare a mutant having a single amino acid substitution for any of the amino acid residues constituting the protein of the present invention.
  • mutants may be screened by any known assay to collect information about the individual mutants, which in turn allows evaluation of the usefulness of individual amino acid residues constituting the protein of the present invention by comparison of the case where a mutant having substitution of a specific amino acid residue shows a lower biological activity than that of the protein of the present invention, the case where such a mutant shows no biological activity, or the case where such a mutant produces unsuitable activity that inhibits the biological activity of the protein of the present invention.
  • those skilled in the art can readily analyze amino acid substitutions undesirable for mutants of the protein of the present invention based on information collected from such routine experiments alone or in combination with other mutations.
  • a protein consisting of an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 can be prepared according to techniques such as site-directed mutagenesis as described in, for example, " Molecular Cloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001 )); “ Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997 ); Kunkel, (1985), Proc. Natl. Acad. Sci. USA, 82: 488-92 ; or Kunkel, (1988), Method Enzymol., 85: 2763-6 ).
  • Preparation of a mutant with such a mutation including amino acid deletion, substitution, or addition may be accomplished, for example, by known procedures such as a Kunkel method or a Gapped duplex method using a mutation-introducing kit based on site-directed mutagenesis such as a QuikChange TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), a GeneTailor TM Site-Directed Mutagenesis System (manufactured by Invitrogen), or a TaKaRa Site-Directed Mutagenesis System (e.g., Mutan-K, Mutan-Super Express Km; manufactured by Takara Bio Inc.).
  • a mutation-introducing kit based on site-directed mutagenesis such as a QuikChange TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), a GeneTailor TM Site-Directed Mutagenesis System (manufactured by Invitrogen), or a TaKaRa Site-
  • Techniques for introducing deletion, substitution, or addition of one or more amino acids in the amino acid sequence of a protein while maintaining its activity include a method of treating a gene with a mutagen and a method selectively cleaving a gene and deleting, substituting, or adding a selected nucleotide and then ligating the gene, in addition to site-directed mutagenesis mentioned above.
  • the nucleotide sequence contained in the nucleic acid of the present invention is preferably a nucleotide sequence that encodes a protein consisting of an amino acid sequence having deletion, substitution, or addition of 1 to 80 amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and having a GPAT activity and/or a GNPAT activity.
  • nucleotide sequence contained in the nucleic acid of the present invention also preferably encompass nucleotide sequences that encode a protein consisting of an amino acid sequence having deletion, substitution, or addition of 1 to 80 amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and having the activity of the present invention.
  • the number and sites of amino acid mutations or modifications in the protein of the present invention are not limited as long as the activity of the present invention is maintained.
  • the activity of the present invention represented by the GPAT activity of the protein, can be measured by a known method, for example, see Biochem. J., 355, 315-322, 2001 .
  • the "GPAT activity" of the present invention may be measured as follows: A microsome fraction is prepared from yeast expressing the GPAT of the present invention by, for example, the method described in J. Bacteriology, 173, 2026-2034 (1991 ) or the like. The microsome fraction is added to a reaction solution containing 0.44 mM glycerol 3-phosphate, 0.36 mM acyl-CoA, 0.5 mM DTT, 1 mg/ml BSA, 2 mM MgCl 2 , and 50 mM Tris-HCl (pH 7.5), followed by reaction at 28°C for an appropriate time. The reaction is terminated by addition of a mixture of chloroform and methanol, and lipids are extracted. The resulting lipids are fractionated by thin layer chromatography or the like to measure the amount of generated lysophosphatidic acid.
  • the activity of the present invention shown in the i), ii), or v) above may be measured by, for example, determining the proportions or contents of fatty acids in a host cell expressing the protein of the present invention (e.g., yeast, M. alpine ).
  • a host cell expressing the protein of the present invention e.g., yeast, M. alpine
  • a mixture of chloroform and methanol adjusted to an appropriate ratio is added to lyophilized cells prepared by a method of producing a fatty acid composition of the present invention, and the resulting mixture is stirred and then heated for an appropriate time.
  • the cells are separated by centrifugation to recover the solvent. This procedure is repeated several times.
  • lipids are dried in an appropriate manner and are then dissolved in a solvent such as chloroform to prepare a sample. From an appropriate amount of this sample, the fatty acids of the cells are converted into methyl ester by a hydrochloric acid-methanol method and are extracted with hexane. Hex
  • the activity of the present invention shown in the iii) above may be measured by, for example, determining the amount of triacylglycerol (TG) of yeast expressing the protein of the present invention.
  • TG triacylglycerol
  • the lipids are extracted and collected from cells as described above, and the TG fraction is collected by fractionation, for example, through thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the fatty acids constituting TG in the collected TG fraction are converted into methyl ester by the hydrochloric acid-methanol method and are extracted with hexane. Hexane is distilled off, followed by gas chromatographic quantitative determination.
  • the activity of the present invention shown in the iv) above may be measured by, for example, confirming whether the introduced protein of the present invention can complement the GPAT deficiency of yeast ( S. cerevisiae ).
  • yeast SCT1 and GPT2 are known as genes involved in the GPAT activity, and it is known that simultaneous deficiency in these genes results in death. That is, yeast deficient in both the SCT1 gene and the GPT2 gene usually cannot grow, but can grow in a complementary manner when a gene having a similar function to these genes, i.e., a protein having a GPAT activity, is expressed.
  • the method for confirming complementation for the GPAT deficiency of yeast may be any method that confirms the recovery of the GPAT activity of yeast strain deficient in the SCT1 gene and the GPT2 gene by expressing the GPAT gene of the present invention.
  • a heterozygous strain in which only one of alleles of the SCT1 gene is deficient is produced.
  • a strain where one expression cassette of the GPAT gene of the present invention is inserted to the heterozygous strain on a chromosome different from the chromosome on which the SCT1 is present or a strain where a plasmid vector having an expression cassette of the GPAT gene of the present invention is inserted to the heterozygous strain is produced.
  • the resulting strain is applied to a spore-forming medium to form ascospores.
  • the resulting cells are subjected to random spore analysis or tetrad analysis to obtain a haploid strain derived from the spores.
  • the genotype of the thus-prepared haploid yeast is inspected.
  • the GPAT of the present invention can be determined to be able to complement a GPAT activity in the yeast.
  • nucleic acid comprising a nucleotide sequence that is hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 under stringent conditions and that encodes a protein having the activity of the present invention
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences that are hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 under stringent conditions and encodes a protein having the activity of the present invention.
  • Such a nucleotide sequence can be prepared from, for example, a cDNA library or a genomic library by a known hybridization technique such as colony hybridization, plaque hybridization, or Southern blotting using a probe produced from an appropriate fragment by a method known to those skilled in the art.
  • a known hybridization technique such as colony hybridization, plaque hybridization, or Southern blotting using a probe produced from an appropriate fragment by a method known to those skilled in the art.
  • hybridization conditions The strength of hybridization conditions is determined primarily based on hybridization conditions, more preferably based on hybridization conditions and washing conditions.
  • stringent conditions used throughout the specification is intended to include moderately or highly stringent conditions.
  • examples of the moderately stringent conditions include hybridization conditions of 1 ⁇ SSC to 6xSSC at 42°C to 55°C, more preferably 1 ⁇ SSC to 3xSSC at 45°C to 50°C, and most preferably 2xSSC at 50°C.
  • a hybridization solution containing, for example, about 50% formamide a hybridization temperature of lower than the temperature mentioned above by 5°C to 15°C is employed.
  • Washing conditions are, for example, 0.5 ⁇ SSC to 6xSSC at 40°C to 60°C.
  • 0.05% to 0.2% SDS preferably about 0.1% SDS, may be usually added.
  • Highly stringent (high stringent) conditions include hybridization and/or washing at higher temperature and/or lower salt concentration, compared to the moderately stringent conditions.
  • the hybridization conditions include 01 ⁇ SSC to 2xSSC at 55°C to 65°C, more preferably 0.1 ⁇ SSC to 1 ⁇ SSC at 60°C to 65°C, and most preferably 0.2 ⁇ SSC at 63°C.
  • Washing conditions are, for example, 0.2 ⁇ SSC to 2xSSC at 50°C to 68°C, and more preferably 0.2xSSC at 60°C to 65°C.
  • hybridization conditions particularly used in the present invention include, but not limited to, prehybridization in 5 ⁇ SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5) and 50% formamide at 42°C, overnight incubation at 42°C in the presence of a probe to form hybrids, and washing in 0.2 ⁇ SSC, 0.1% SDS at 65°C for 20 minutes three times.
  • a commercially available hybridization kit not using radioactive substance as a probe.
  • a DIG nucleic acid detection kit (Roche Diagnostics) or an ECL direct labeling & detection system (manufactured by Amersham) is used for hybridization.
  • nucleotide sequence falling within the present invention include nucleotide sequences that are hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 under conditions of 2 ⁇ SSC at 50°C and encode a protein having a GPAT activity and/or a GNPAT activity.
  • nucleic acid comprising a nucleotide sequence that consists of a nucleotide sequence having an identity of 70% or more with the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 and encodes a protein having the activity of the present invention
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences that have an identity of at least 70% with the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 and encode a protein having the activity of the present invention.
  • a nucleic acid comprises a nucleotide sequence having an identity of at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98%, or 99% or more) with the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 and encoding a protein having the activity of the present invention.
  • the percent identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation, but is preferably determined by comparing sequence information of two nucleic acids using a computer program.
  • sequence comparison for example, the BLASTN program ( Altschul et al., (1990), J. Mol. Biol., 215: 403-10 ) version 2.2.7, available via the National Library of Medicine website: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html or the WU-BLAST 2.0 algorithm can be used. Standard default parameter settings for WU-BLAST 2.0 are described at the following Internet site: http://blast.wustl.edu.
  • nucleic acid comprising a nucleotide sequence encoding an amino acid sequence having an identity of 70% or more with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and encoding a protein having the activity of the present invention
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences encoding an amino acid sequence having an identity of 70% or more with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and encoding a protein having the activity of the present invention.
  • the protein encoded by the nucleic acid of the present invention may be any protein having an identity with the amino acid sequence of MaGPAT4, MaGPAT4-long, or MaGPAT5 as long as the protein is functionally equivalent to the protein having the activity of the present invention.
  • the protein include amino acid sequences having an identity of 75% or more, preferably 80% or more, more preferably 85% or more, and most preferably 90% or more (e.g., 95% or more, furthermore 98% or more) with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9.
  • the nucleotide sequence contained in the nucleic acid of the present invention is preferably a nucleotide sequence encoding an amino acid sequence having an identity of 90% or more with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and encoding a protein having the activity of the present invention.
  • the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation or can be determined using a computer program.
  • a computer program examples include BLAST, FASTA ( Altschul et al., J. Mol. Biol., 215: 403-410, (1990 )) and ClustalW.
  • various conditions (parameters) for an identity search with the BLAST program are described by Altschul et al. (Nucl. Acids. Res., 25, pp.
  • a specific alignment scheme for aligning a plurality of amino acid sequences can show matching of sequences also in a specific short region and can therefore detect a region having a very high sequence identity in such a short region even if the full-length sequences have no significant relationship therebetween.
  • the BLAST algorithm can use the BLOSUM62 amino acid scoring matrix, and the following selection parameters can be used: (A) inclusion of filters to mask a segment of a query sequence having low compositional complexity (as determined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993); also see Wootton and Federhen, 1996, "Analysis of compositionally biased regions in sequence databases", Methods Enzymol., 266: 554-71 ) or to mask segments consisting of short-periodicity internal repeats (as determined by the XNU program of Claverie and States (Computers and Chemistry, 1993), and (B) a statistical significance threshold for reporting matches against database sequences, or the expected probability of matches being found merely by chance, according to the statistical model of E-score (Karlin and Altschul, 1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.
  • E-score Kerlin and Altschul, 1990
  • nucleic acid comprising a nucleotide sequence that is hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 under stringent conditions and encodes a protein having an activity of the present invention
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences that are hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 under stringent conditions and encode a protein having an activity of the present invention.
  • the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and the hybridization conditions are as described above.
  • Examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences that are hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 under stringent conditions and encode a protein having the activity of the present invention.
  • nucleic acid comprising a nucleotide sequence that is hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 under stringent conditions and includes an exon encoding a protein having the activity of the present invention
  • the nucleotide sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 12 are the genomic DNA sequences encoding MaGPAT4 (and MaGPAT4-long) and MaGPAT5, respectively, of the present invention.
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences that are hybridizable with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 under stringent conditions and include an exon encoding a protein having the activity of the present invention.
  • nucleotide sequence can be prepared by a method known to those skilled in the art from, for example, a genomic library by a known hybridization technique such as colony hybridization, plaque hybridization, or Southern blotting using a probe produced using an appropriate fragment.
  • the hybridization conditions are as described above.
  • nucleic acid comprising a nucleotide sequence that consists of a nucleotide sequence having an identity of 70% or more with the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 and includes an exon encoding a protein having the activity of the present invention
  • nucleotide sequence contained in the nucleic acid of the present invention encompass nucleotide sequences that have an identity of at least 70% with the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 and encode a protein having the activity of the present invention.
  • nucleotide sequence examples include those having an identity of at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98%, or 99% or more) with the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 and having an exon encoding a protein having the activity of the present invention.
  • the percent identity between two nucleotide sequences can be determined as described above.
  • the genomic DNA sequence set forth in SEQ ID NO: 7 is composed of ten exons and nine introns.
  • the exon regions correspond to nucleotides 428 to 744 or 596 to 744, 850 to 924,1302 to 1396, 1480 to 1726, 1854 to 2279, 2370 to 2632, 2724 to 3299, 3390 to 3471, 3575 to 4024, and 4133 to 4248.
  • the genomic DNA sequence set forth in SEQ ID NO: 12 is composed of three exons and two introns. In SEQ ID NO: 12, the exon regions correspond to nucleotides 1 to 302, 457 to 1676, and 1754 to 2598.
  • examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences including intron regions having a nucleotide sequence identity of 100% with the genomic DNA sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 and exon regions having a nucleotide sequence identity of at least 70% or more, more preferably 75% or more, and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98%, or 99% or more) with the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12, wherein the exon encodes a protein having the activity of the present invention.
  • examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences including exon regions having a nucleotide sequence identity of 100% with the genomic DNA sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 and intron regions having a nucleotide sequence identity of at least 70% or more, more preferably 75% or more, and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98%, or 99% or more) with the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12, wherein the intron regions can be eliminated by splicing, and thereby the exon regions are ligated to encode a protein having the activity of the present invention.
  • examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences including intron regions having a nucleotide sequence identity of at least 70% or more, more preferably 75% or more, and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98%, or 99% or more) with the genomic DNA sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 and exon regions having a nucleotide sequence identity of at least 70% or more, more preferably 75% or more, and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95% or more, 98% or more, or 99% or more) with the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12, wherein the intron regions can be eliminated by splicing, and thereby the exon regions are ligated to encode a protein having the activity of the present
  • the percent identity between two nucleotide sequences can be determined by the method described above.
  • nucleic acid of the present invention encompass nucleic acids each consisting of a nucleotide sequence having deletion, substitution, or addition of one or more nucleotides in the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 and encoding a protein having the activity of the present invention. More specifically, a usable nucleic acid includes any one of the following nucleotide sequences:
  • nucleic acid of the present invention also encompasses nucleic acids comprising a fragment of a nucleotide sequence shown in any one of (a) to (d) below:
  • nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8 (b)the nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9, and (c) the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 11 are as shown in Table 1.
  • the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12 is also as described above.
  • the fragments of these sequences include ORF, CDS, a biologically active region, a region used as a primer as described later, or a region which may serve as a probe contained in these nucleotide sequences, and may be either naturally occurring or artificially prepared.
  • nucleic acid of the present invention encompass the following nucleic acids.
  • Glycerol 3-phosphate acyltransferase of the present invention Glycerol 3-phosphate acyltransferase of the present invention
  • proteins of the present invention encompass a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 and proteins functionally equivalent to such a protein. These proteins may be either naturally occurring or artificially prepared.
  • the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 is as described above.
  • the "proteins functionally equivalent” refers to proteins having "the activity of the present invention," described in the "Nucleic acid encoding glycerol 3-phosphate acyltransferase of the present invention" above.
  • proteins functionally equivalent to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 include proteins according to any one of (a) and (b) below:
  • amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 or the amino acid sequence having an identity of 70% or more with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9 are as described in the "Nucleic acid encoding glycerol 3-phosphate acyltransferase of the present invention" above.
  • the "protein having the activity of the present invention” encompasses mutants of proteins encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8; mutated proteins by many types of modification such as deletion, substitution, and addition of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 9; modified proteins having, for example, modified amino acid side chains; and fused proteins with other proteins, where these proteins have the GPAT activity, the GNPAT activity, and/or the activity i) or the activity ii) described in the "Nucleic acid encoding glycerol 3-phosphate acyltransferase of the present invention" above.
  • the protein of the present invention may be artificially prepared.
  • the protein can be produced by chemical synthesis such as a Fmoc method (fluorenylmethyloxycarbonyl method) or a tBoc method (t-butyloxycarbonyl method).
  • Fmoc method fluorenylmethyloxycarbonyl method
  • tBoc method t-butyloxycarbonyl method
  • peptide synthesizers available from Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, or other manufacturers may be used for chemical synthesis.
  • Examples of the protein of the present invention further encompass the following proteins.
  • the GPAT nucleic acid of the present invention can be cloned by, for example, screening from a cDNA library using an appropriate probe.
  • the cloning can be performed by PCR amplification using appropriate primers and subsequent ligation to an appropriate vector.
  • the cloned nucleic acid may be further subcloned into another vector.
  • plasmid vectors such as pBlue-Script TM SK(+) (Stratagene), pGEM-T (Promega), pAmp (TM: Gibco-BRL), p-Direct (Clontech), or pCR2.1-TOPO (Invitrogen), can be used.
  • a primer may be any region of, e.g., the nucleotide sequence set forth in SEQ ID NO: 3, 6, or 11.
  • primers described in Examples shown below can be used.
  • PCR is performed using cDNA prepared from M. alpina cells with the primers above, DNA polymerase, and any other substance.
  • PCR conditions in the present invention may be, for example, as follows:
  • the resulting PCR product can be purified by a known method, for example, using a kit such as GENECLEAN kit (Funakoshi Co., Ltd.), QIAquick PCR purification (QIAGEN), or ExoSAP-IT (GE Healthcare Bio-Sciences); a DEAE-cellulose filter; or a dialysis tube.
  • a kit such as GENECLEAN kit (Funakoshi Co., Ltd.), QIAquick PCR purification (QIAGEN), or ExoSAP-IT (GE Healthcare Bio-Sciences); a DEAE-cellulose filter; or a dialysis tube.
  • the PCR product is subjected to agarose gel electrophoresis, and nucleotide sequence fragments are cut out from the agarose gel and are purified, for example, with a GENECLEAN kit (Funakoshi Co., Ltd.) or a QIAquick Gel extraction kit (QIAGEN) or by a freeze-squeeze method.
  • the nucleotide sequence of the cloned nucleic acid can be determined with a nucleotide sequencer.
  • the present invention also provides a recombinant vector containing a nucleic acid encoding the GPAT of the present invention.
  • the present invention further provides a transformant transformed with such a recombinant vector.
  • the recombinant vector and transformant can be prepared as follows: A plasmid having a nucleic acid encoding the GPAT of the present invention is digested with a restriction enzyme. Examples of the restriction enzyme include, but not limited to, EcoRI, KpnI, BamHI, and SalI. The end may be blunted with T4 polymerase. A digested DNA fragment is purified by agarose gel electrophoresis. This DNA fragment is incorporated into an expression vector by a known method in order to prepare a vector for GPAT expression. This expression vector is introduced into a host to prepare a transformant, which is provided for expression of a desired protein.
  • the expression vector and the host may be any types that allow expression of a desired protein.
  • the host include fungi, bacteria, plants, animals, and cells thereof.
  • fungi include filamentous fungi such as lipid-producing M. alpina and yeast strains such as Saccharomyces cerevisiae.
  • bacteria include Escherichia coli and Bacillus subtilis.
  • plants include oil plants such as rapeseed, soybean, cotton, safflower, and flax.
  • microorganisms belonging to the genus Mortierella such as microorganisms belonging to subgenus Mortierella, e.g., Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFOS94f, Mortierella alpina IFO8568, ATCC16266, ATCC32221, ATCC42430, CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS528.72, CBS529.72, CBS608.70, and CBS754.68; and microorganisms belonging to subgenus Micromucor, e.g., Mortierella isabellina CBS194.28, IFO6336, IFO7824, IFO7873, IFO7874,
  • the nucleic acid of the present invention is preferably self-replicable in the host or preferably has a structure insertable onto the fungal chromosome.
  • the nucleic acid also includes a promoter and a terminator.
  • M. alpina is used as a host, for example, pD4, pDuraSC, or pDura5 can be used as the expression vector.
  • Any promoter that allows expression in the host can be used, and examples thereof include promoters derived from M. alpina, such as histonH4.1 gene promoter, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene promoter, and TEF (translation elongation factor) gene promoter.
  • Examples of the method introducing a recombinant vector into filamentous fungi such as M. alpina include electroporation, a spheroplast method, a particle delivery method, and direct microinjection of DNA into nuclei.
  • the transformed strain can be obtained by selecting a strain that grows on a selective medium lacking a certain nutrient(s).
  • a colony of drug-resistant cells can be obtained by culturing the host cells in a selective medium containing the drug.
  • yeast When yeast is used as a host, for example, pYE22m can be used as the expression vector.
  • yeast expression vectors such as pYES (Invitrogen) or pESC (Stratagene) may be used.
  • yeast expression vectors such as pYES (Invitrogen) or pESC (Stratagene) may be used.
  • yeast expression vectors such as pYES (Invitrogen) or pESC (Stratagene) may be used.
  • Examples of the host suitable for the present invention include, but not limited to, Saccharomyces cerevisiae strain EH13-15 (trp1, MAT ⁇ ).
  • the promoter that can be used is, for example, a promoter derived from yeast, such as GAPDH promoter, gall promoter, or gal10 promoter.
  • Examples of the method introducing a recombinant vector into yeast include a lithium acetate method, electroporation, a spheroplast method, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into nuclei.
  • a bacterium such as E . coli
  • pGEX or pUC18 available from Pharmacia can be used as the expression vector.
  • the promoter that can be used includes those derived from, for example, E. coli or phage, such as trp promoter, lac promoter, PL promoter, and PR promoter. Examples of the method of introducing a recombinant vector into bacteria include electroporation and calcium chloride methods.
  • the present invention provides a method of preparing a fatty acid composition from the transformant described above, i.e., a method of preparing a fatty acid composition from a cultured product obtained by culturing the transformant.
  • the fatty acid composition contains an assembly of one or more fatty acids therein.
  • the fatty acids may be free fatty acids or may be present in the form of lipids containing fatty acids, such as triglyceride or phospholipid.
  • the fatty acid composition of the present invention can be prepared by the following method.
  • the fatty acid composition can also be prepared by any other known method.
  • the medium used for culturing an organism expressing GPAT may be any culture solution (medium) that has an appropriate pH and osmotic pressure and contains biomaterials such as nutrients necessary for growth of each host, trace elements, serum, and antibiotics.
  • medium in the case of expressing GPAT by transforming yeast, unlimited examples of the medium include SC-Trp medium, YPD medium, and YPD5 medium.
  • composition of a specific medium is as follows:
  • One liter of the medium includes 6.7 g of yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g of leucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, and 0.6 g of uracil).
  • DIFCO yeast nitrogen base w/o amino acids
  • amino acid powder a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid,
  • Any culture conditions which are suitable for host growth and adequate for stably maintaining the generated enzyme may be employed. Specifically, individual conditions including anaerobic degree, culture period, temperature, humidity, and static culture or shake culture can be adjusted. Culture may be accomplished under the same conditions (one-step culture) or by so-called two-step or three-step culture using two or more different culture conditions. For large-scale culture, two- or more-step culture is preferred because of its high culture efficiency.
  • the fatty acid composition of the present invention can be prepared as follows: As pre-culture, a colony of a transformant is inoculated in, for example, the SC-Trp medium and shake-cultured at 30°C for 2 days. Subsequently, as main culture, 500 ⁇ L of the pre-culture solution is added to 10 mL of YPD5 (2% yeast extract, 1% polypeptone, and 5% glucose) medium, followed by shake culture at 30°C for 2 days.
  • YPD5 2% yeast extract, 1% polypeptone, and 5% glucose
  • the present invention also provides a fatty acid composition as an assembly of one or more fatty acids in cells expressing the GPAT of the present invention, preferably, a fatty acid composition obtained by culturing a transformant expressing the GPAT of the present invention.
  • the fatty acids may be free fatty acids or may be present in the form of lipids containing fatty acids, such as triglyceride or phospholipid.
  • the fatty acids contained in the fatty acid composition of the present invention are linear or branched monocarboxylic acids of long-chain carbohydrates, and examples thereof include, but not limited to, myristic acid (tetradecanoic acid) (14:0), myristoleic acid (tetradecenoic acid) (14:1), palmitic acid (hexadecanoic acid) (16:0), palmitoleic acid (9-hexadecenoic acid) (16:1), stearic acid (octadecanoic acid) (18:0), oleic acid (cis-9-octadecenoic acid) (18:1(9)), vaccenic acid (11-octadecenoic acid) (18:1(11)), linolic acid (cis,cis-9,12 octadecadienoic acid) (18:2(9,12)), ⁇ -linolenic acid (9,12,15-octadecatrienoic acid) (18:
  • the fatty acid composition of the present invention may be composed of any number and any type of fatty acids, as long as it is a combination of one or more fatty acids selected from the fatty acids mentioned above.
  • the proportions of fatty acids in the fatty acid composition of the present invention can be determined by the method of determining the compositional ratio or the contents of fatty acids described in the "Nucleic acid encoding glycerol 3-phosphate acyltransferase of the present invention" in the specification.
  • the present invention also provides a food product comprising the fatty acid composition described above.
  • the fatty acid composition of the present invention can be used for, for example, production of food products containing fats and oils and production of industrial raw materials (for example, raw materials for cosmetics, pharmaceuticals (e.g., external applications for the skin), and soaps), in usual methods.
  • Cosmetics (cosmetic compositions) or pharmaceuticals (pharmaceutical compositions) may be formulated into any dosage form including, but not limited to, solutions, pastes, gels, solids, and powders.
  • Examples of the forms of food products include pharmaceutical formulations such as capsules; natural liquid diets, semi-digested nutritious diets, and elemental nutritious diets where the fatty acid composition of the present invention is blended with proteins, sugars, fats, trace elements, vitamins, emulsifiers, and flavorings; and processed forms such as drinkable preparations and enteral nutrients.
  • examples of the food product of the present invention include, but not limited to, nutritional supplements, health food, functional food, children's food, modified milk for infants, modified milk for premature infant, and geriatric food.
  • the term "food” is used as a collective term for edible materials in the form of a solid, a fluid, a liquid, or a mixture thereof.
  • the term "nutritional supplements” refers to food products enriched with specific nutritional ingredients.
  • health food refers to food products that are healthful or good for health and encompasses nutritional supplements, natural food, and diet food.
  • functional food refers to food products for supplying nutritional ingredients that assist body control functions and is synonymous with food for specified health use.
  • children's food refers to food products given to children up to about 6 years old.
  • geriatric food refers to food products treated to facilitate digestion and absorption thereof, compared to untreated food.
  • modified milk for infants refers to modified milk given to children up to about one year old.
  • modified milk for premature infants refers to modified milk given to premature infants until about 6 months after birth.
  • Examples of these food products include natural food (treated with fats and oils) such as meat, fish, and nuts; food supplemented with fats and oils during preparation, such as Chinese foods, Chinese noodles, and soups; food products prepared using fats and oils as heating media, such as tempura (deep-fried fish and vegetables), deep-fried food, fried tofu, Chinese fried rice, doughnuts, and Japanese fried dough cookies (karinto)); fat- and oil-based food or processed food supplemented with fats and oils during processing, such as butter, margarine, mayonnaise, dressing, chocolate, instant noodles, caramel, biscuits, cookies, cake, and ice cream; and food sprayed or coated with fats and oils upon finishing, such as rice crackers, hard biscuits, and sweet bean paste bread.
  • natural food treated with fats and oils
  • food supplemented with fats and oils during preparation such as Chinese foods, Chinese noodles, and soups
  • food products prepared using fats and oils as heating media such as tempura (deep-fried fish and vegetables), deep-fried food,
  • the food products of the present invention are not limited to food containing fats and oils, and other examples thereof include agricultural food products such as bakery products, noodles, cooked rice, sweets (e.g., candies, chewing gums, gummies, tablets, Japanese sweets), tofu, and processed products thereof; fermented food products such as refined sake, medicinal liquor, seasoning liquor (mirin), vinegar, soy sauce, and miso; livestock food products such as yogurt, ham, bacon, and sausage; seafood products such as fish paste (kamaboko), deep-fried fish paste (ageten), and fish cake (hanpen); and fruit drinks, soft drinks, sports drinks, alcoholic beverages, and tea.
  • agricultural food products such as bakery products, noodles, cooked rice, sweets (e.g., candies, chewing gums, gummies, tablets, Japanese sweets), tofu, and processed products thereof; fermented food products such as refined sake, medicinal liquor, seasoning liquor (mirin), vinegar, soy sauce, and miso; livestock food products such as yogurt, ham, bacon
  • the present invention also provides a method of evaluating or selecting a lipid-producing fungus using the GPAT-encoding nucleic acid or GPAT protein of the present invention. Details are given below.
  • One embodiment of the present invention is a method of evaluating a lipid-producing fungus using the GPAT-encoding nucleic acid or GPAT protein of the present invention.
  • a lipid-producing fungus strain as a test strain is evaluated for the activity of the present invention using primers or probes designed based on the nucleotide sequence of the present invention.
  • Such evaluation can be performed by known procedures, for example, described in International Publication No. WO01/040514 and JP-A-8-205900 . The method for evaluation will be briefly described below.
  • the first step is preparation of a genome of a test strain.
  • the genome can be prepared by any known method such as a Hereford method or a potassium acetate method (see, e.g., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, p. 130 (1990 )).
  • Primers or probes are designed based on the nucleotide sequence of the present invention, preferably the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8. These primers or probes may be any regions of the nucleotide sequence of the present invention and may be designed by a known procedure.
  • the number of nucleotides in a polynucleotide used as a primer is generally 10 or more, preferably 15 to 25.
  • the number of nucleotides appropriate for a region to be flanked by primers is generally 300 to 2000.
  • the primers or probes prepared above are used to examine whether the genome of a test strain contains a sequence specific to the nucleotide sequence of the present invention or not.
  • the sequence specific to the nucleotide sequence of the present invention can be detected by a known procedure.
  • a polynucleotide containing a part or all of the sequence specific to the nucleotide sequence of the present invention or a polynucleotide containing a nucleotide sequence complementary to the nucleotide sequence is used as one primer, and a polynucleotide containing a part or all of a sequence located upstream or downstream of this sequence or a polynucleotide containing a nucleotide sequence complementary to the nucleotide sequence is used as the other primer, and a nucleic acid from the test strain is amplified by PCR or other techniques. Further, for example, the presence or absence of an amplification product and the molecular weight of an amplification product can be measured.
  • PCR conditions suitable for the method of the present invention are not particularly limited and may be, for example, as follows:
  • a test strain can be evaluated for the activity of the present invention by culturing the test strain and measuring the expression level of GPAT encoded by the nucleotide sequence of the present invention, e.g., the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6.
  • the expression level of GPAT can be measured by culturing a test strain under appropriate conditions and quantifying mRNA or protein for GPAT.
  • the mRNA or protein can be quantified by a known procedure. For example, mRNA can be quantified by Northern hybridization or quantitative RT-PCR, and protein can be quantified by Western blotting ( Current Protocols in Molecular Biology, John Wiley & Sons, 1994-2003 ).
  • Another embodiment of the present invention is a method of selecting a lipid-producing fungus using the GPAT-encoding nucleic acid or GPAT protein of the present invention.
  • a strain having a desired activity can be selected by culturing a test strain, measuring the expression level of GPAT encoded by the nucleotide sequence of the present invention, e.g., the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 8, and selecting a strain of a desired expression level.
  • a desired strain can be selected by establishing a standard strain, culturing the standard strain and a test strain separately, measuring the expression level of each strain, and comparing the expression level of the standard strain with that of the test strain.
  • a standard strain and test strains are cultured under appropriate conditions, and the expression level of each strain is measured.
  • a strain exhibiting a desired activity can be selected by selecting a test strain showing higher or lower expression than the standard strain does.
  • the desired activity can be determined by, for example, measuring the expression level of GPAT and the composition of fatty acids produced by GPAT, as described above.
  • a test strain having a desired activity can also be selected by culturing test strains and selecting a strain having high or low activity of the present invention.
  • a desired activity can be determined by, for example, measuring the expression level of GPAT and the composition of fatty acids produced by GPAT, as described above.
  • test strain and the standard strain include, but not limited to, strains transformed with the vector of the present invention, strains modified to suppress expression of the nucleic acid of the present invention, mutagenized strains, and naturally mutated strains.
  • the activity of the present invention can be measured by, for example, the method described in the "Nucleic acid encoding glycerol 3-phosphate acyltransferase of the present invention" in the specification.
  • mutagenesis examples include, but not limited to, physical methods such as irradiation with ultraviolet light or radiation; and chemical methods by treatment with a chemical such as EMS (ethylmethane sulfonate) or N-methyl-N-nitrosoguanidine (see, e.g., Yasuji Oshima ed., Biochemistry Experiments vol. 39, Experimental Protocols for Yeast Molecular Genetics, pp. 67-75, Japan Scientific Societies Press ).
  • EMS ethylmethane sulfonate
  • N-methyl-N-nitrosoguanidine see, e.g., Yasuji Oshima ed., Biochemistry Experiments vol. 39, Experimental Protocols for Yeast Molecular Genetics, pp. 67-75, Japan Scientific Societies Press ).
  • strain used as the standard strain of the present invention or the test strain include, but not limited to, the lipid-producing fungus and yeast described above.
  • the standard strain and the test strain may be any combination of strains belonging to different genera or species, and one or more test strains may be simultaneously used.
  • M. alpina strain 1S-4 was inoculated into 100 mL of a GY2:1 medium (2% glucose, 1% yeast extract, pH 6.0) and was shake-cultured at 28°C for 2 days. The cells were collected by filtration and genomic DNA was prepared by using DNeasy (QIAGEN).
  • the nucleotide sequence of the genomic DNA was determined with a Roche 454 GS FLX Standard. On this occasion, the nucleotide of a fragment library was sequenced in two runs, and the nucleotide of a mate pair library was sequenced in three runs. The resulting nucleotide sequences were assembled to obtain 300 supercontigs.
  • M. alpina strain 1S-4 was inoculated into 4 mL of a medium (2% glucose, 1% yeast extract, pH 6.0) and was cultured at 28°C for 4 days. The cells were collected by filtration, and RNA was extracted with an RNeasy plant kit (QIAGEN). Complementary DNA was synthesized using a SuperScript First-Strand system for RT-PCR (Invitrogen). In addition, from the total RNA, poly(A) + RNA was purified using an Oligotex-dT3Q ⁇ Super>mRNA Purification Kit (Takara Bio Inc.). A cDNA library was constructed with a ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene).
  • the amino acid sequence of GPAT derived from yeast ( S. cerevisiae ), SCT1 (YBL011W) or GPT2 (YKR067W) was subjected to tblastn search for M. alpha strain 1S-4 genomic nucleotide sequences. As a result, supercontigs including the sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 gave hits.
  • SEQ ID NO: 13 was the genomic sequence of MaGPAT1
  • SEQ ID NO: 15 was the genomic sequence of MaGPAT2 ( WO2008/156026 ).
  • SEQ ID NO: 14 was the genomic sequence of MaGPAT3, which has been separately identified before by the present inventors (unpublished at the time of filing of the present application).
  • the genes relating to SEQ ID NO: 7 and SEQ ID NO: 12 were believed to be novel.
  • the gene relating to SEQ ID NO: 7 was named MaGPAT4, and the gene relating to SEQ ID NO: 12 was named MaGPAT5.
  • a nucleotide sequence of a supercontig comprising the sequence set forth in SEQ ID NO: 4 was subjected to BLAST analysis and was compared with a known GPAT homolog. The result suggested that TAA at the 4246 to 4248 positions in the sequence set forth in SEQ ID NO: 4 was the stop codon. The start codon was difficult to be presumed from comparison with known homologs. Accordingly, cloning of the 5'-end of cDNA was attempted by a 5'-RACE method. That is, the cDNA on the 5' side upstream than the following primer:
  • PCR was performed with ExTaq (Takara Bio Inc.) using the cDNA as a template and a combination of primer SacI-GPAT4-1 and primer Sal-GPAT4-2 at 94°C for 2 min and then 30 cycles of (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min).
  • the amplified DNA fragment of about 2.5 kbp was cloned with a TOPO-TA cloning Kit (Invitrogen).
  • a nucleotide sequence of the insert region was determined, and a plasmid having a nucleotide sequence set forth in SEQ ID NO: 3 was named pCR-MaGPAT4.
  • a nucleotide sequence of a CDS encoding MaGPAT4 is shown in SEQ ID NO: 3
  • a nucleotide sequence of the ORF is shown in SEQ ID NO: 1
  • an amino acid sequence of MaGPAT4 deduced from these nucleotide sequences is shown in SEQ ID NO: 2.
  • MaGPAT4-long The sequence in the case of using the ATG at the 428 to 430 positions as a start codon was defined as MaGPAT4-long.
  • a nucleotide sequence of a CDS encoding MaGPAT4-long is shown in SEQ ID NO: 6
  • a nucleotide sequence of the ORF is shown in SEQ ID NO: 4
  • an amino acid sequence of MaGPAT4-long deduced from these nucleotide sequences is shown in SEQ ID NO: 5.
  • PCR was performed with ExTaq (Takara Bio Inc.) using the thus-constructed library as a template and a combination of primer GPAT5-1F and primer GPAT5-3R at 94°C for 2 min and then 30 cycles of (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min).
  • the resulting DNA fragment of about 0.9 kbp was cloned with a TOPO-TA cloning Kit (Invitrogen).
  • a nucleotide sequence of the insert region was determined, and a plasmid having a nucleotide sequence of the 1503 to 2385 positions of SEQ ID NO: 8 was named pCR-MaGPAT5-P.
  • probes was produced by PCR using these plasmids as templates and the primers in the above.
  • ExTaq (Takara Bio Inc., Japan) was used, except that a PCR labeling mix (Roche Diagnostics) was used instead of the attached dNTP mix for labeling DNA to be amplified with digoxigenin (DIG) to prepare MaGPAT5 probes.
  • DIG digoxigenin
  • Hybridization conditions were set as follows:
  • a DIG nucleic acid detection kit (Roche Diagnostics) was used for detection. Plasmids were cut out by in vivo excision from phage clones obtained by screening to obtain each plasmid DNA. A plasmid having the longest insert among the plasmids obtained by screening with the MaGPAT5 probe included the nucleotide sequence set forth in SEQ ID NO: 11 and was named plasmid pB-MaGPAT5. The nucleotide sequence set forth in SEQ ID NO: 11 was searched for ORF. As a result, a CDS having a start codon at the 225 to 227 positions of SEQ ID NO: 11 and a stop codon at the 2589 to 2591 positions of SEQ ID NO: 11 were found.
  • the genomic sequence (SEQ ID NO: 7) and the CDS sequence (SEQ ID NO: 3) of the MaGPAT4 gene were compared with each other. The result suggested that the genomic sequence of this gene is composed of ten exons and nine introns and encodes a protein consisting of 825 amino acid residues ( Figures 1 and 2 ). Comparison between the genomic sequence (SEQ ID NO: 12) and the CDS sequence (SEQ ID NO: 10) of the MaGPAT5 gene suggested that the genomic sequence of this gene is composed of three exons and two introns and encodes a protein consisting of 788 amino acid residues ( Figures 3 and 4 ).
  • MaGPAT4 and MaGPAT5 were compared with a known GPAT homolog derived from Mortierella alpina.
  • Tables 2 and 3 show CDS sequences and identity of amino acid sequences deduced from the CDS sequences.
  • Table 2 Identity (%) of CDS with GPAT homolog derived from Mortierella MaGPAT1 MaGPAT2 MaGPAT3 MaGPAT4 MaGPAT5 MaGPAT1 - 42.4 73.2 44.9 44.5 MaGPAT2 - 38.7 39.9 39.0 MaGPAT3 - 45.1 45.5 MaGPAT4 - 44.0 MaGPAT5 -
  • Table 3 Identity (%) of amino acid sequence with GPAT homolog derived from Mortierella MaGPAT1 MaGPAT2 MaGPAT3 MaGPAT4 MaGPAT5 MaGPAT1 - 16.0 83.7 13.1 18.7 MaGPAT2 - 14.4 13.8 11.6 MaGPAT3 - 13.5 18.0 MaGPAT4 - 11.3 MaGPAT5 -
  • the deduced amino acid sequence (SEQ ID NO: 2) of MaGPAT4 was subjected to homology analysis against amino acid sequences registered in GenBank nr with BLASTp.
  • the amino acid sequence showing the lowest E-value against this sequence, i.e., having the highest identity was the amino acid sequence (GenBank Accession No. XP_001224211) of a deduced protein derived from ascomycete Chaetomium globosum CBS 148.51, and the identity thereof was 39.3%.
  • the amino acid sequence also had a homology with glycerone phosphate O-acyltransferase (GNPAT) derived from an animal, and the identity with human GNPAT (GenBank Accession No. AAH00450) was 22.6%.
  • the deduced amino acid sequence (SEQ ID NO: 9) of MaGPAT5 was subjected to homology analysis against amino acid sequences registered in GenBank nr with BLASTp.
  • the amino acid sequence showing the lowest E-value against this sequence, i.e., having the highest identity was the amino acid sequence (GenBank accession No. XP_759516) of a deduced protein UM03369 derived from ascomycete Ustilago maydis 521, and the identity thereof was 15.4%.
  • Figure 6 shows alignment of MaGPAT5 with these amino acid sequences.
  • MaGPAT4 and MaGPAT5 conserved the region that is conserved in acyltransferase, in particular, conserved the glycine residue (G) and the proline residue (P) indicated by the symbol *, which are considered to be important for the acyltransferase activity. Furthermore, the serine residue (S) indicated by the symbol +, which is considered to be important for binding with G3P, was also conserved ( Figures 5 and 6 ).
  • a DNA fragment prepared by digesting yeast expression vector pYE22m ( Biosci. Biotech. Biochem., 59, 1221-1228, 1995 ) with restriction enzymes EcoRI and SalI and a DNA fragment of about 2.1 kbp prepared by digesting plasmid pCR-MaGPAT4 with restriction enzymes EcoRI and SalI were linked each other by using ligation high (TOYOBO) to construct plasmid pYE-MaGPAT4.
  • MaGPAT1, MaGPAT2, or MaGPAT3 in yeast were constructed for comparison.
  • the vectors expressing MaGPAT1 and MaGPAT2 in yeast were constructed as described in WO2008/156026 , and named pYE-MaGPAT1 and pYE-MaGPAT2, respectively.
  • the vector expressing MaGPAT3 in yeast was prepared as follows. A tblastn search was performed against M. alpina strain 1S-4 genomic nucleotide sequences prepared as in Example 1 using the amino acid sequence of MaGPAT1 (ATCC No. 16266) as a query. As a result, a gene having a homology with MaGPAT1 was identified and was named MaGPAT3 (unpublished at the time of filing of the present application).
  • Yeast S . cerevisiae strain EH13-15 (trp1, MAT ⁇ ) ( Appl. Microbiol. Biotechnol., 30, 515-520, 1989 ) was transformed by a lithium acetate method using plasmid pYE22m, pYE-MaGPAT4, pYE-MaGPAT1, pYE-MaGPAT2, or pYE-MaGPAT3.
  • Transformants that grew on SC-Trp (containing, per liter, 6.7 g of yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g of leucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, and 0.6 g of uracil)) agar medium (2% agar) were selected.
  • amino acid powder a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid,
  • arbitrary two strains from the strains obtained by transformation with plasmid pYE-MaGPAT1 were named MaGPAT1-1 strain and MaGPAT1-2 strain
  • arbitrary two strains from the strains obtained by transformation with plasmid pYE-MaGPAT2 were named MaGPAT2-1 strain and MaGPAT2-2 strain
  • arbitrary two strains from the strains obtained by transformation with plasmid pYE-MaGPAT3 were named MaGPAT3-1 strain and MaGPAT3-2 strain
  • these strains were also subjected to the following culturing experiment as in the MaGPAT4-1 strain and MaGPAT4-2 strain.
  • one platinum loop of yeast cells from the plate were inoculated in 10 mL of an SC-Trp medium and were shake-cultured at 30°C for 1 day.
  • 500 ⁇ L of the pre-culture solution was added to 10 mL of an SC-Trp medium or a YPD (2% yeast extract, 1% polypeptone, and 2% glucose) medium, followed by shake culture at 30°C for 2 days.
  • the yeast cells were collected by centrifugation of the culture solution.. The cells were washed with 10 mL of sterilized water, collected again by centrifugation, and lyophilized. To the lyophilized yeast cells, 4 mL of a mixture containing chloroform : methanol (2:1) was added and vigorously agitated, and then allowed to stand at 70°C for 1 hour. The yeast cells were separated by centrifugation, and the solvent was collected. To the remaining cells, 4 mL of the mixture of chloroform : methanol (2:1) was added again, and the solvent was collected in a similar manner. The lipid was dried with Speedback, and 2 mL of chloroform was added thereto to dissolve the lipid.
  • Table 5 Fatty acid composition (%) in cells (Medium: SD-Trp) C-1 C-2 MaGPAT4-1 MaGPAT4-2 MaGPAT2-1 MaGPAT2-2 16:0 7.22 7.51 19.94 21.97 7.19 7.08 16:1 36.71 36.49 16.18 17.41 32.66 33.02 18:0 5.87 5.98 11.66 7.03 6.41 6.67 18:1 45.90 46.17 47.86 48.43 49.25 49.15 Others 4.29 3.85 4.35 5.17 4.48 4.09
  • Table 6 Fatty acid content (%) in cells C-1 C-2 MaGPAT4-1 MaGPAT4-2 MaGPAT2-1 MaGPAT2-2 2.89 2.89 5.32 5.73 2.95 4.30 (Medium: YPD)
  • Table 7 Fatty acid content (%) in cells C-1 C-2 MaGPAT4-1 MaGPAT4-2 MaGPAT2-1 MaGPAT2-2 5.36 4.86 9.04 11.63 5.93 5.89 (Medium: SD-Trp)
  • the compositional ratio of fatty acids constituting the lipid in the cells of a strain highly expressing MaGPAT4 has a high ratio of palmitic acid (16:0) and a low ratio of palmitoleic acid (16:1), compared with those of the control strain into which the vector only was introduced.
  • This tendency is different from the tendencies when another GPAT derived from Mortierella alpina, i.e., MaGPAT1, MaGPAT2, or MaGPAT3, was used.
  • MaGPAT4 of the present invention has a substrate specificity different from those of other GPATs derived from Mortierella alpina.
  • a plasmid containing a galactose-inducible promoter was constructed as follows.
  • PCR was performed with ExTaq (Takara Bio Inc.) using the cDNA prepared in Example 2 as a template and a combination of primer Not-MaGPAT4-F1 and primer Bam-MaGPAT4-R or a combination of primer Not-MaGPAT4-F2 and primer Bam-MaGPAT4-R at 94°C for 2 min and then 30 cycles of (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min).
  • the amplified DNA fragments of about 2.7 kbp and about 2.5 kbp were cloned with a TOPO-TA cloning Kit (Invitrogen) and were confirmed to have the CDS sequence of GPAT4-long set forth in SEQ ID NO: 6 and the CDS sequence of GPAT4 set forth in SEQ ID NO: 3, respectively, and named plasmid pCR-MaGPAT4-long-1 and plasmid pCR-MaGPAT4-1, respectively.
  • the DNA fragment of about 6.5 kbp obtained by digestion of vector pESC-TRP (Stratagene) with restriction enzymes NotI and BglII was linked to a DNA fragment of about 2.7 kbp or 2.5 kbp obtained by digestion of plasmid pCR-MaGPAT4-long-1 or plasmid pCR-MaGPAT4-1 with restriction enzymes NotI and BamHI using ligation high (TOYOBO) to prepare plasmid pESC-T-MaGPAT4-long or plasmid pESC-T-MaGPAT4.
  • Yeast S . cerevisiae strain EH13-15 was transformed by a lithium acetate method using pESC-TRP, pESC-T-MaGPAT4-long, or pESC-T-MaGPAT4. Transformants that grew on an SC-Trp agar medium were selected.
  • the yeast cells were collected by centrifugation of the culture solution. The cells were washed with 10 mL of sterilized water, collected again by centrifugation, and lyophilized. The fatty acids in the cells of one line of each strain were converted into methyl ester by a hydrochloric acid-methanol method and were subjected to gas chromatographic analysis of the total fatty acids contained in the cells (Table 8).
  • Lipids in the cells of another line of each strain were extracted as follows. That is, 1 mL of a mixture containing chloroform : methanol (2:1) and glass beads were added to the cells, and the cells were disrupted with a bead beater. Thereafter, centrifugation was conducted and the supernatant was collected. Further one milliliter of the mixture of chloroform : methanol (2:1) was added to the remaining cells, and the supernatant was similarly collected. This procedure was repeated, and lipids were extracted with 4 mL of the mixture of chloroform : methanol (2:1) in total. The solvent was distilled off with Speedback, and the residue was dissolved in a small amount of chloroform.
  • the lipids were visualized by spraying a primulin solution and then irradiated with UV light.
  • the triacylglycerol (TG) fraction, free-fatty acid (FFA) fraction, diacylglycerol (DG) fraction, and phospholipid (PL) fraction were scraped from the plate and were respectively put in tubes.
  • the fatty acids were converted into methyl esters by a hydrochloric acid-methanol method and were subjected to gas chromatographic analysis of the fatty acids (Table 9, Figure 7 ).
  • Table 8 Total amount of fatty acids in cells (SG-Trp culture) Control MaGPAT4-long MaGPAT4 147.12 ⁇ 6.18 254.58 ⁇ 6.16 290.08 ⁇ 19.67 Average ⁇ SD
  • Table 9 Amount of fatty acids in lipid fraction (SG-Trp culture) Control MaGPAT4-long MaGPAT4 FFA 5.12 ⁇ 1.26 8.53 ⁇ 1.37 13.98 ⁇ 2.80 TG 43.18 ⁇ 2.76 122.55 ⁇ 4.40 149.74 ⁇ 16.81 DG 4.75 ⁇ 0.38 10.78 ⁇ 0.28 22.08 ⁇ 5.15 PL 87.09 ⁇ 2.49 96.03 ⁇ 2.88 103.88 ⁇ 8.02 Average ⁇ SD
  • the total amounts of fatty acids in the strain expressing MaGPAT4-long and in the strain expressing MaGPAT4 increased by 1.7-fold and 2.0-fold, respectively, compared with that in the control (Table 8).
  • the amounts of PL in the strain expressing MaGPAT4-long and in the strain expressing MaGPAT4 were respectively 1.1-fold and 1.2-fold that in the control. Thus, these strains showed almost no difference to the control.
  • the amounts of TG in the strain expressing MaGPAT4-long and in the strain expressing MaGPAT4 notably increased to be 2.8-fold and 3.5-fold, respectively, that in the control. That is, expression of MaGPAT4-long or MaGPAT4 could activate biosynthesis of fatty acids and enhance the productivity of a reserve lipid, TG.
  • the amounts of DG and FFA in the strain expressing MaGPAT4-long and in the strain expressing MAGPAT2 increased compared with those in the control (Table 9).
  • the proportions of fatty acids in each lipid fraction are shown in Figure 7 .
  • the proportions of fatty acids in the strain expressing MaGPAT4-long and in the strain expressing MaGPAT4 did almost not differ from those in the control, whereas, in the other fractions, there were tendency of an increase in saturated fatty acids and a decrease in unsaturated fatty acids in the strain expressing MaGPAT4-long and in the strain expressing MaGPAT4.
  • 16:0 (palmitic acid) increased
  • 16:1 palmitoleic acid
  • the main culture was performed using an SC-Trp liquid medium instead of the SG-Trp liquid medium. Since the SC-Trp liquid medium does not contain galactose, expression of MaGPAT4-long or MaGPAT4 introduced by transformation is not induced in the main culture using this medium. In the experiment using the SC-Trp liquid medium, no differences were observed in both the amount and proportions of the total fatty acids of the cells between the control and the strain introduced with MaGPAT4-long or MaGPAT4 (Table 10, Figure 8 ). This also suggested that expression of MaGPAT4-long or MAGPAT2 was induced by galactose, resulting in an increase of the fatty acids in the cells in the SG-Trp medium.
  • Table 10 Total amount of fatty acids in cells (SC-Trp culture) Control GPAT4-long GPAT4 mg/L broth 153.26 ⁇ 3.05 150.98 ⁇ 6.48 156.35 ⁇ 8.06
  • Example 7 Complementary experiment of yeast S. cerevisiae ⁇ sct1 and ⁇ gpt2
  • SCT1 and GPT2 are known as genes involved in the GPAT activity, and simultaneous deficiency in these genes is known to result in death.
  • SCT1 and GPT2 are known as genes involved in the GPAT activity, and simultaneous deficiency in these genes is known to result in death.
  • a complementary experiment of ⁇ sct1 and ⁇ gpt2 was performed. Table 11 summarizes the genotypes of strains produced as below.
  • the SCT1 gene of ⁇ gpt2 homozygous diploid yeast (catalog No. YSC1021-663938) of a yeast knock out strain collection (Open Biosystems) was disrupted as follows. DNA was extracted from S. cerevisiae strain S288C cells using Dr. GenTLE (from yeast) (TaKaRa Bio Inc.).
  • a fragment of the SCT1 gene was amplified by PCR with KOD-Plus-(TOYOBO) using the resulting DNA as a template, primer Xba1-Des-SCT1-F: 5'-TCTAGAATGCCTGCACCAAAACTCAC-3' (SEQ ID NO: 31) and primer Xba1-Des-SCT1-R: 5'-TCTAGACCACAAGGTGATCAGGAAGA-3' (SEQ ID NO: 32).
  • the amplified DNA fragment of about 1.3 kbp was cloned using a Zero Blunt TOPO PCR cloning kit (Invitrogen), and the resulting plasmid was named pCR-SCT1P.
  • a DNA fragment of about 2.2 kbp containing CDS of the LEU2 gene obtained by digestion of plasmid YEp13 with restriction enzymes Sail and XhoI was linked to a DNA fragment of about 4.4 kbp obtained by digestion of plasmid pCR-SCT1P with SalI using ligation high (TOYOBO) to prepare a plasmid, pCR- ⁇ sct1:LEU2, where the LEU2 gene was inserted in reverse orientation with respect to the SCT1 gene.
  • a transformant that grew on SD-Leu (containing, per liter, 6.7 g of yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, and 0.6 g of uracil)) agar medium (2% agar) was selected.
  • amino acid powder a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid,
  • DNA was extracted from the resulting transformant cells by the method described above. PCR was performed using a combination (a) primer SCT1outORF-F: 5'-AGTGTAGGAAGCCCGGAATT-3' (SEQ ID NO: 33) and primer SCT1inORF-R: 5'-GCGTAGATCCAACAGACTAC-3' (SEQ ID NO: 34) (0.5 kbp) or a combination (b) primer SCT1outORF-F and primer LEU2inORF-F: 5'-TTGCCTCTTCCAAGAGCACA-3' (SEQ ID NO: 35) (1.2 kbp) to confirm the genotype, i.e., to confirm to be SCT1/ ⁇ sct1:LEU2, and the strain was named GP-1 strain.
  • a DNA fragment of about 6.6 kbp obtained by digestion of vector pESC-URA (Stratagene) with restriction enzymes NotI and BglII was linked to a DNA fragment of about 2.7 kbp or 2.5 kbp obtained by digestion of plasmid pCR-MaGPAT4-long-1 or plasmid pCR-MaGPAT4-1 with restriction enzymes NotI and BamHI using ligation high (TOYOBO) to prepare plasmid pESC-U-MaGPAT4-long and plasmid pESC-U-MaGPAT4.
  • Yeast GP-1 strain was transformed by a lithium acetate method using pESC-URA, pESC-U-MaGPAT4-long, or pESC-U-MaGPAT4. Transformants that grew on an SC-Ura (containing, per liter, 6.7 g of yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, and 1.8 g of leucine)) agar medium
  • the strain obtained by transformation with pESC-URA was named C-D strain
  • strains obtained by transformation with pESC-U-MaGPAT4-long and pESC-U-MaGPAT4 were named MaGPAT4-long-D strain and MaGPAT4-D strain, respectively.
  • the C-D strain, MaGPAT4-long-D strain, and MaGPAT4-D strain were each applied to a YPD agar medium and were cultured at 30°C for 1 day.
  • the grown cells were applied to a spore-forming agar medium (0.5% potassium acetate, 2% agar) and were cultured at 20°C for 7 days.
  • An appropriate amount of the resulting cells were scraped and were suspended in 100 ⁇ L of a zymolyase solution (0.125 mg/mL of zymolyase 100T, 1M sorbitol, 40 mM potassium phosphate buffer (pH 6.8)).
  • the suspension was incubated at room temperature for 30 min, and the tube containing the suspension was then placed in ice.
  • the resulting spore clones were replicated by incubation on an SG-Ura (containing, per liter, 6.7 g of yeast nitrogen base w/o amino acids (DIFCO), 20 g of galactose, and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, and 1.8 g of leucine)) agar medium (2% agar) and an SG-Leu (containing, per liter, 6.7 g of yeast nitrogen base w/o amino acids (D
  • the genotypes of the resulting strains were investigated by extracting DNA from the cells as described above and performing PCR using a combination (a) primer SCT1outORF-F and primer SCT1inORF-R and a combination (b) primer SCT1outORF-F and primer LEU2in ORF-F.
  • the leucine non-auxotrophic strains were not amplified by the PCR using the combination (a), but were amplified by the PCR using the combination (b). These strains were thus confirmed to have ⁇ sct1:LEU2 alleles.
  • the leucine auxotrophic strains were amplified by the PCR using the combination (a), but were not amplified by the PCR using the combination (b). These strains were thus confirmed to have SCT1 alleles.
  • strains derived from the four ascospores isolated from MaGPAT4-long-D strain or MaGPAT4-D strain were each applied onto a YPD agar medium. The growth of two strains was well, but the growth of the other two strains was considerably poor. The strains showing poor growth corresponded to the leucine non-auxotrophic strains having ⁇ sct1:LEU2 alleles.
  • vectors were constructed as follows.
  • Vector pUC18 was digested with restriction enzymes EcoRI and HindIII, and an adapter prepared by annealing oligoDNAs, MCS-for-pUC18-F2 and MCS-for-pUC18-R2 was inserted to construct plasmid pUC18-RF2.
  • Primer URA5g-F1 5'-GTCGACCATGACAAGTTTGC-3' (SEQ ID NO: 40)
  • Primer URA5g-R1 5'-GTCGACTGGAAGACGAGCACG-3' (SEQ ID NO: 41)
  • a DNA fragment of about 1.0 kbp amplified by PCR with KOD-plus-(TOYOBO) using the genome of M. alpina as a template and primer hisHp+URA5-F and primer hisHp+MGt-F was linked to a DNA fragment of about 5.3 kbp amplified by PCR with KOD-plus- (TOYOBO) using pDuraG as a template and primer pDuraSC-GAPt-F and primer URA5gDNA-F using an In-Fusion (registered trademark) Advantage PCR Cloning Kit (TaKaRa Bio Inc.) to prepare plasmid pDUra-RhG.
  • Primer hisHp+URA5-F 5'-GGCAAACTTGTCATGAAGCGAAAGAGAGATTATGAAAACAAGC-3' (SEQ ID NO: 42)
  • Primer hisHp+MGt-F 5'-CACTCCCTTTTCTTAATTGTTGAGAGAGTGTTGGGTGAGAGT-3' (SEQ ID NO: 43)
  • Primer pDuraSC-GAPt-F 5'-TAAGAAAAGGGAGTGAATCGCATAGGG-3' (SEQ ID NO: 44)
  • Primer URA5gDNA-F 5'-CATGACAAGTTTGCCAAGATGCG-3' (SEQ ID NO: 45)
  • a DNA fragment of about 6.3 kbp was amplified by PCR with KOD-plus-(TOYOBO) using plasmid pDUra-RhG as a template and primer pDuraSC-GAPt-F and primer pDurahG-hisp-R.
  • Primer pDurahG-hisp-R 5'-ATTGTTGAGAGAGTGTTGGGTGAGAGTG-3' (SEQ ID NO: 46)
  • a DNA fragment of about 2.5 kbp was amplified by PCR with KOD-plus-(TOYOBO) using plasmid pCR-MaGPAT4-1 as a template and primer MaGPAT4+hisp-F and primer MaGPAT4+MGt-R.
  • Primer MaGPAT4+hisp-F 5'-CACTCTCTCAACAATATGACAACCGGCGACAGTACCGC-3' (SEQ ID NO: 47)
  • Primer MaGPAT4+MGt-R 5'-CACTCCCTTTTCTTATTATAATTTCGGGGCGCCATCGC-3' (SEQ ID NO: 48)
  • the resulting fragment of 2.5 kbp was linked to the above-mentioned DNA fragment of 6.3 kbp using an In-Fusion (registered trademark) Advantage PCR Cloning Kit (TaKaRa Bio Inc.) to prepare plasmid pDUraRhG-GPAT4.
  • Transformation was performed using a uracil auxotrophic strain ⁇ ura-3 induced from M. alpina strain 1S-4 in accordance with the method described in Patent Literature ( WO2005/019437 , Title of Invention: "Method of breeding lipid-producing fungus") as a host by a particle delivery method using plasmid pDUraRhG-GPAT4.
  • Transformants were selected using an SC agar medium (0.5% yeast nitrogen base w/o amino acids and ammonium sulfate (Difco), 0.17% ammonium sulfate, 2% glucose, 0.002% adenine, 0.003% tyrosine, 0.0001% methionine, 0.0002% arginine, 0.0002% histidine, 0.0004% lysine, 0.0004% tryptophan, 0.0005% threonine, 0.0006% isoleucine, 0.0006% leucine, 0.0006% phenylalanine, 2% agar).
  • SC agar medium 0.5% yeast nitrogen base w/o amino acids and ammonium sulfate (Difco), 0.17% ammonium sulfate, 2% glucose, 0.002% adenine, 0.003% tyrosine, 0.0001% methionine, 0.0002% arginine, 0.0002% histidine, 0.0004% lysine, 0.0004% tryptophan
  • RT-PCR was performed using a combination of the following primers:
  • strains confirmed of overexpression was inoculated in 10 mL of a GY medium (2% glucose, 1% yeast extract) and shake-cultured at 28°C at 300 rpm for 3 days.
  • the whole culture solution was added to 500 mL of a GY medium (2-L Sakaguchi flask) and shake-cultured at 28°C at 120 rpm.
  • Five milliliters and ten milliliters of the culture solution were sampled on the third, seventh, tenth, and twelfth days and were filtered.
  • the cells were dried at 120°C.
  • Fatty acids were converted into methyl esters by a hydrochloric acid-methanol method and were subjected to gas chromatographic analysis of the fatty acids.
  • a change with time in amount of arachidonic acid produced per dry cells was investigated.
  • the host for transformation, ⁇ ura-3 strain, was used as a control. The results are shown in Figure 9 .
  • the amount of arachidonic acid (AA) per cells increased in M. alpina overexpressing GPAT4 compared with that in the control.

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